A fundamental question in liver biology is how type I cannabinoid receptors (CB1Rs) mediate liver fibrosis. Cannabinoid receptors are a class of G-protein coupled receptors (GPCRs). Cannabinoid receptors exhibit marginal expression in the normal liver but undergo enhanced expression in the fibrotic human liver, predominately in activated hepatic stellate cells (HSCs)2. A critical aspect in chronic liver disease is the activation of resident HSCs into proliferative, contractile, and fibrogenic cells. These myofibroblasts produce an excess of extracellular matrix proteins including collagens, resulting in fibrosis. In these cells, specific mitogenic signaling cascades, upon activation of naturally occurring GPCRs such as AT1R and adenosine 2A receptor (A2aR) contribute to the fibrosis. HSCs are the cells primarily responsible for the fibrogenic response in the liver1. Preventing the expression or activation of CB1Rs attenuates the development of fibrosis2. CB1Rs contribute to fibrogenic response, since administration of the CB1R antagonist Rimonabant (SR141716) or genetic ablation of CB1R inhibits fibrosis progression in three distinct models of chronic liver injury (namely, CCl4-, thioacetamide-, and bile duct ligation-induced fibrosis). However, the extent of the contribution of CB1Rs in alcohol-induced liver injury, and the molecular mechanism by which CB1Rs promote liver fibrosis are not understood.
Approximately 25,000 people die each year of cirrhosis in the United States. The leading causes of cirrhosis are viral hepatitis, alcoholism and obesity-related liver diseases. Close to 1 million people suffer from this disease, which is the 7th leading cause of death in the United States, and whose only effective treatment is liver transplantation. It is known that cannabinoid receptors are upregulated in the liver during liver fibrosis, and the blockade of the CB1 subtype of the receptor has been shown to inhibit fibrogenesis, offering an approach for the treatment of cirrhosis. However, as CB1R expression is not localized specifically to the liver (in fact, CB1Rs are likely the most widely expressed GPCR in the brain), the treatment of liver fibrosis and cirrhosis by the administration of a CB1R inhibitor would like result in significant side effects. In fact, the targeting of CB1 receptor in the treatment of obesity has posed serious safety problems associated with severe side effects. For example, (1) the Sanofi-Aventis CB1R antagonist Rimonabant (SR141716) was taken off the market because of its serious side effects; (2) Merck discontinued clinical trials with its CB1R inverse agonist Taranabant (MK-0364) due to the high level of central side effects, mainly depression and anxiety; and (3) Bristol Myers Squibb also discontinued development of its CB1R antagonist Otenabant (CP-945,598) following the problems seen during clinical use of the similar drug Rimonabant. Therefore, what are needed are specific antagonists that do not have these systemic, toxic side effects.
N-arachidonoyl-ethanolamine (AEA, anandamide) and 2-arachidonoyl glycerol (2-AG) are the two most studied endocannabinoids. They are biosynthesized by cleavage of their membrane lipid precursors N-arachidonoyl-phosphatidylethanolamine and sn-1-acyl-2-arachidonoylglycerols (DAGs) respectively, and then inactivated by intracellular hydrolyzing enzymes. The biological activity of AEA and 2-AG is mainly mediated by activation of the cannabinoid receptors CB1R and CB2R.
2-AG has a variety of effects in vivo; it is a mediator of neurite outgrowth, during brain development, or as retrograde signal mediating depolarization-induced suppression of neurotransmission and synaptic plasticity, in the adult brain. It protects neurons from inflammation by preventing Cox2 gene expression (D2). In addition, 2-AG is involved in metabolic regulation and diseases. For example, it is involved in bone formation (D3), in obesity (its concentration is increased in several tissues and in the circulation of obese persons (D4)). 2-AG is highly upregulated during chronic liver diseases and is implicated in the pathogenesis of non-alcoholic fatty liver disease, progression of fibrosis to cirrhosis and the development of the cardiovascular abnormalities of cirrhosis, such as the hyperdynamic circulatory syndrome and cirrhotic cardiomiopathy (D5).
2-AG is produced from the hydrolysis of phosphoinositol bisphosphate (PIP2), catalyzed by the PIP2-selective phospholipase C, or from the hydrolysis of phosphatidic acid (PA), catalyzed by a PA phosphohydrolase into diacylglycerols (DAGs). DAGs are then converted into 2-AG by sn-1 selective-DAG lipases (DAGLs). Two sn-1 DAG lipase isozymes (DAGLα and DAGLβ) have been cloned and enzymatically characterized (D6). They are mostly located in the plasma membrane, are stimulated by Ca2+ and glutathione, appear to possess a catalytic triad typical of serine hydrolases, and do not exhibit strong selectivity for 2-arachidonate-containing DAGs (FIG. 1). Endocannabinoids, including 2-AG, stimulate CB1R, resulting, among other things, in the activation of the ERK1/2 pathway, and phosphorylation of ERK1/2.
Because AEA and 2-AG biosynthetic enzymes have been identified only recently, little information on the development of selective inhibitors for these proteins is currently available. RHC 80267 [1,6-bis-(cyclohexyloximino-carbonylamino)-hexane], and tetrahydrolipstatin (THL), have been shown to inhibit DAGL at concentrations lower than those required to inhibit other lipases (D6). Furthermore, two inhibitors of 2-AG biosynthesis have been developed so far, O-3640 and O-3841, with excellent selectivity for DAGLα over the other proteins of the endocannabinoid system tested. However, they are not suitable for in vivo use (D7). Therefore, the identification of new inhibitors of DAGLs, which could be used in vivo, is highly clinically relevant.